This invention is in the field of molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding scorpion toxins that are potassium channel agonists.
Scorpion venoms have been recognized as a source of peptidyl inhibitors of various types of potassium ion (K) channels. Some of these peptides have been purified to homogeneity and their properties characterized. The most extensively studied of these toxins is charybdotoxin (ChTX). ChTX is a thirty-seven amino acid peptide isolated from venom of the old world scorpion Leiurus quinquestriatus var. hebraeus. Originally described as an inhibitor of the high-conductance, Ca+2-activated K (Maxi-K) channel present in muscle and neuro-endocrine cells, ChTX was later found also to inhibit a number of different medium-and small-conductance Ca+2-activated K-channels, as well as a voltage-dependent K-channel (K(v) 1.3). In each case, channel inhibition occurs with similar potency, in the low nanomolar range. A related toxin, iberiotoxin (IbTX), shares 68% sequence homology with ChTX and selectively blocks the Maxi-K channel. Other peptidyl inhibitors, such as limbatustoxin (LbTX) and kaliotoxin (KTX), have also been shown to possess greater selectivity for the Maxi-K channel. Other peptidyl toxins homologous to ChTX have been identified (e.g., noxiustoxin).
Potassium channels modulate a number of cellular events such as muscle contraction, neuro-endocrine secretion, frequency and duration of action potentials, electrolyte homeostasis, and resting membrane potential. These channels comprise a family of proteins that have been classified according to their biophysical and pharmacological characteristics. Inhibition of K-channels, in their role as modulators of the plasma membrane potential in human T-lymphocytes, has been postulated to play a role in eliciting immunosuppressive responses. In reggulating membrane potential, K-channels play a role in the regulation of intracellular Ca+2 homeostasis, which has been found to be important in T-cell activation.
Potassium channel agonists are small polypeptides (31 to 37 amino acids) which form compact structures kept rigid by three disulfide bridges. Use of synthetic analogs with point mutations has determined that single amino acids residues are important for receptor binding and for biological activity of K-channel toxins (Sabatier et al. (1994) Int. J. Peptide Protein Res. 43:486495). Moreover, a drug with high affinity for the receptor could be expected to produce irreversible blockade of synaptic transmission. When labeled with a tracer molecule, such a drug would provide a reliable way of tagging receptors to permit measurement of their number and distribution within cells and tissues. These features would have very valuable consequences for research on excitatory amino acid neurotransmission and for the development of therapeutic agents to treat central nervous system dysfunction in humans and animals. Methods for treating heart and neurological diseases by applying toxins derived from spiders have been described (U.S. Pat. No. 4,925,664).
Arthropod animals, including insects, and certain parasitic worms, use excitatory amino acids as a major chemical neurotransmitter at their neuromuscular junction and in their central nervous system. Because of the damage done by insect pests and the prevalence of parasitic worm infections in animals and humans in many countries, there is a constant need for potent and specific new pesticides and anthelmintic drugs that are non-toxic to humans, pets, and farm animals.
Many arthropods produce a mixture of insecticidal proteins referred to as venom. These toxic substances are synthesized in specialized glandular tissues, which, when directed by a stinging or piercing apparatus, are capable of paralyzing the arthropod""s prey. Small, slow moving or stationary arthropods have adapted a strategy to instantaneously paralyze their prey by utilizing neurotoxic components of the venom at very low concentrations. These components or neurotoxins interfere with the function of insect nervous tissues through efficient competition for certain receptor sites. Many of these neurotoxins are polypeptides. These have been divided into different classes based on their host specificity and mode of action (Zlotkin (1991) Phytoparasitica 19:177-182). For example, neurotoxic peptides isolated from numerous species of scorpions have been divided into classes that affect arthropods and classes that affect mammals.
Due to a combination of problems associated with some synthetic insecticides, including toxicity, environmental hazards, and loss of efficacy due to resistance, there exists a continuing need for the development of novel means of invertebrate control, including the development of genetically engineered recombinant baculoviruses which express protein toxins capable of incapacitating the host more rapidly than the baculovirus infection per se.
Scorpion venoms have been identified as possible sources of compounds providing insecticidal properties. Two insect-selective toxins isolated from the venom of the scorpion Leiurus quinquestriatus and affecting sodium conductance have been reported previously (Zlotkin et al. (1985) Arch. Biochem. and Biophysics 240:877-87). One toxin, AaIT, induced fast excitatory contractive paralysis of fly larvae and the other, LqhIT2, induced slow depressant flaccid paralysis suggesting that these two toxins have different chemical and pharmacological properties (Zlotkin et al. (1971) Biochimie (Paris) 53:1073-1078). Thus, other toxins derived from scorpion venom will also have different chemical and pharmacological properties.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a K-channel agonist polypeptide having at least 25 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a scorpion K-channel agonist polypeptide of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, and 15 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as an insect, a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2 polypeptide having at least 25 amino acids and at least 95% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, and 16.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2 polypeptide in a host cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level a potassium channel blocking toxin 15- 1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2 polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin.2 polypeptide in. the host cell containing the isolated polynucleotide with the level of a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2 polypeptide in a host cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2 polypeptide gene, preferably a scorpion K-channel agonist polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2 amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a subsantial portion of the amino acid sequence encoding a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2 polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
Another embodiment of the instant invention pertains to a method for expressing a gene encoding a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2 in the genome of a recombinant baculovirus in insect cell culture or in viable insects wherein said insect cells or insects have been genetically engineered to express a potassium channel blocking toxin 15-1, an agitoxin 1, a leiuropeptide II, a kaliotoxin 2 precursor, a tityustoxin k alpha, a charybdotoxin, or a charybdotoxin 2.